CN117363565A - Construction method of vascularized stem cell sphere for bone regeneration - Google Patents
Construction method of vascularized stem cell sphere for bone regeneration Download PDFInfo
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- CN117363565A CN117363565A CN202311279958.4A CN202311279958A CN117363565A CN 117363565 A CN117363565 A CN 117363565A CN 202311279958 A CN202311279958 A CN 202311279958A CN 117363565 A CN117363565 A CN 117363565A
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Abstract
The invention belongs to the technical field of bone tissue engineering, and discloses a construction method of vascularized stem cell spheres for bone regeneration. In particular discloses a stem cell sphere with osteogenic and angiogenic differentiation, which is a gingival mesenchymal stem cell (STRO 1) positive by STRO1 + GMSCs, sGMSCs) and Human Umbilical Vein Endothelial Cells (HUVECs) were obtained by three-dimensional co-culture. The vascularized stem cell sphere provided by the invention consists of gingival mesenchymal stem cells (sGMSC) expressing STRO1s) and Human Umbilical Vein Endothelial Cells (HUVECs). The vascularized stem cell pellet can not only highly express the markers of bone formation and blood vessel formation, such as ALP, RUNX2, OCN, CD31, VEGF and the like, but also solve the defect of single stem cell pellet in hematopoietic differentiation and realize vascularization and bone tissue regeneration of the stem cell pellet.
Description
Technical Field
The invention belongs to the technical field of bone tissue engineering, and particularly relates to a construction method of vascularized stem cell spheres for bone regeneration.
Background
Trauma, tumors, inflammation can lead to various degrees of bone tissue loss or loss, and bone tissue regeneration is dependent on abundant blood circulation. Due to the lack of angiogenesis, the conventional bone tissue engineering material has low field planting and differentiation potential of the transplanted seed cells on the scaffold material, and finally affects the regeneration and reconstruction efficiency of new bone.
The stem cell ball is a three-dimensional structure micro-organ which is cultivated in vitro based on the self-aggregation and self-assembly characteristics of adult stem cells, and is a potential bone tissue engineering material due to the functional characteristics similar to the tissue or organ from which the stem cells are derived in vivo.
Although the stem cell pellet is hopeful to be a good bone tissue engineering material, the stem cell pellet is completely composed of stem cells, lacks complex structures such as blood vessels and the like, and is easy to necrose in early transplantation, so that the application of the stem cell pellet in transplantation and clinical transformation is limited. Human Umbilical Vein Endothelial Cells (HUVECs) that highly express hematopoietic lineage CD31 provide the potential for vascularization of mesenchymal stem cell-derived stem cell pellets.
The Gingival Mesenchymal Stem Cells (GMSCs) are derived from neural crest in early embryo development, have strong immunoregulation property and multi-lineage differentiation effect, and compared with bone marrow mesenchymal stem cells (BMSCs), adipose mesenchymal stem cells (ADSCs) and other seed cells commonly used for bone tissue engineering, the GMSCs have stronger in vitro osteogenic differentiation capability.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a vascularized stem cell sphere for bone tissue engineering, which effectively solves the problems of lack of vascular network, early necrosis after transplantation, low bone regeneration efficiency and the like of the stem cell sphere in the research and application of the bone tissue engineering at the present stage.
The object of the first aspect of the present invention is to provide a stem cell pellet having osteogenic and angiogenic differentiation.
The object of the second aspect of the present invention is to provide a method for preparing the stem cell pellet of the first aspect of the present invention.
The object of the third aspect of the present invention is to provide the use of the stem cell pellet of the first aspect of the present invention or the method of preparation of the second aspect of the present invention.
The object of the fourth aspect of the present invention is to provide a vascularized tissue.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a stem cell pellet having osteogenic and angiogenic differentiation, the stem cell pellet being derived from STRO 1-positive gingival mesenchymal stem cells (STRO 1) + GMSCs, sGMSCs) and HUVECs are obtained by three-dimensional co-culture.
In some embodiments of the invention, the sGMSCs highly express the mesenchymal stem cell markers CD73, CD90 and CD105, and highly express the progenitor cell marker STRO1.
Primary GMSCs isolated from tissues are not single cell lines and there may be differences in the level of stem in the cell population. Progenitor cell marker STRO1 is one of the important manifestations of the high and low level of cell stem, and Mesenchymal Stem Cells (MSCs) highly expressing STRO1 generally have higher osteogenic differentiation potential. Thus by enriching and selecting a subset of GMSCs (STRO 1 + GMSCs, sGMSCs) are of great significance for improving the regeneration potential and the therapeutic effect.
The vascularized stem cell sphere constructed by three-dimensional co-culture of sGMSCs and HUVECs can keep the phenotype stable in vitro, the introduction of the HUVECs promotes the whole cell activity of the stem cell sphere based on the sGMSCs, promotes the expression of an osteogenic differentiation marker, overcomes the defect of single stem cell sphere in the aspect of angiogenesis, and can form a network vessel with a receiving area after transplantation to realize bone tissue regeneration.
At present, a single stem cell which is cultured in two dimensions is generally adopted as a seed cell applied to bone tissue engineering, and the seed cell is proliferated and osteogenic differentiated on a bracket material so as to realize the repair of bone tissue defects; compared to two-dimensional culture induction, three-dimensional cultured stem cells can self-assemble into spheres, better mimic the interactions between cell-cell and cell-extracellular matrix (ECM), and thus exhibit superior cell biological behaviors such as higher cellular activity, phenotypic stability, differentiation potential, and protein secretion function.
Since most mesenchymal stem cells for bone tissue engineering lack the capacity of hemangiogenic differentiation, the transplanted seed cells are liable to lack vascular networks to supply nutrition and oxygen, which is unfavorable for early survival and regeneration potential activation of the transplanted cells. Umbilical Vein Endothelial Cells (HUVECs) are capable of highly expressing angiogenic factors such as CD31, CD34, VEGF, etc., and proved to promote MSCs cell activity and differentiation potential. HUVECs introduction of co-culture constructed vascularized stem cell pellets (i.e., sGMSCs/HUVECs spheres, GHS) may help to make up for the lack of stem cell pellets (i.e., sGMSCs spheres, GS) in terms of angiogenesis and may form networked vessels with the recipient area after implantation.
In some embodiments of the invention, the ratio of sGMSCs to HUVECs is (1-6): 1.
In some preferred embodiments of the invention, the ratio of sGMSCs to HUVECs is (1-5): 1.
In some preferred embodiments of the invention, the ratio of sGMSCs to HUVECs is 1:1.
In some embodiments of the invention, the stem cells highly express the vascularization markers CD31 and VEGF, and highly express the osteogenic differentiation markers RUNX2 and OCN.
In some embodiments of the invention, the stem cell pellet has a diameter of 500 to 700 μm.
In some preferred embodiments of the invention, the stem cell pellet has a diameter of 500 to 600 μm.
In some embodiments of the invention, the method for screening sGMSCs comprises:
after washing GMSCs by DPBS and digestion by pancreatin, re-suspending by buffer solution, mixing with buffer solution containing PE coupled rabbit anti-human STRO1 antibody, incubating for 10-15 min in dark, and sorting to obtain sGMSCs.
In some embodiments of the invention, the sorting comprises sorting by fluorescence activated cell sorting, magnetic cell sorting, fluorometry, flow cytometry, or microscopy.
In some embodiments of the invention, observation under a microscope, osteogenic differentiation alizarin red S staining, and adipogenic differentiation oil red O staining may be used to verify whether sGMSCs are obtained.
In a second aspect of the present invention, there is provided a method for preparing a stem cell pellet of the first aspect of the present invention, comprising the steps of: and mixing sGMSCs and HUVECs, and culturing in an induction medium to obtain stem cell pellets.
In some embodiments of the invention, the sGMSCs and HUVECs are mixed in a cell ratio of (1-6): 1.
In some embodiments of the invention, the sGMSCs and HUVECs are mixed in a cell ratio of (1-5): 1.
In some preferred embodiments of the invention, the sGMSCs and HUVECs are mixed in a cell ratio of 1:1.
In some embodiments of the invention, the induction medium comprises EGM2, alpha-MEM, FBS, glutaMAX, L-ascorbic acid, dexamethasone, and sodium beta-glycerophosphate.
In some embodiments of the invention, the volume ratio of the alpha-MEM to EGM2 in the induction medium is (1-6): 1.
In some embodiments of the invention, the volume ratio of the alpha-MEM to EGM2 in the induction medium is (1-5): 1.
In some preferred embodiments of the invention, the volume ratio of the alpha-MEM to EGM2 in the induction medium is 1:1.
In some embodiments of the invention, the induction medium comprises EGM2, alpha-MEM, 1% -2%P/S+8% -12% FBS+1% -2% Glutamax+90-120 mu M L-ascorbic acid+8-15 nM dexamethasone+8-16 mM sodium beta-glycerophosphate.
In some embodiments of the invention, the time of the culturing is 1 to 14 days.
In some embodiments of the invention, the time of the culturing is 1 to 7 days.
In some preferred embodiments of the invention, the incubation time is 1 to 5 days.
In some embodiments of the invention, the method further comprises gelling by mixing a gel (e.g., gelMA hydrogel, matrigel, type I collagen hydrogel, etc. scaffold material) with the stem cell pellet.
In a third aspect of the invention there is provided the use of the stem cell pellet of the first aspect of the invention or the method of preparation of the second aspect of the invention in any one of (a 1) to (a 6):
(a1) Preparing vascularized tissue;
(a2) Preparing a product for promoting bone tissue repair/regeneration;
(a3) Preparing an angiogenesis promoting product;
(a4) Screening for anti-angiogenic/pro-angiogenic drugs;
(a5) The cell activity of stem cells in vitro induction is improved;
(a6) Prolonging the cell activity time of stem cells in vitro.
In a fourth aspect of the invention there is provided vascularised tissue comprising stem cell pellets of the first aspect of the invention subjected to induction culture.
The beneficial effects of the invention are as follows:
the stem cell sphere with osteogenic and angiogenic differentiation provided by the invention is obtained by three-dimensional co-culture of sGMSCs and HUVECs. The vascularized stem cell pellet can not only highly express the markers of bone formation and blood vessel formation, such as ALP, RUNX2, OCN, CD31, VEGF and the like, but also solve the defect of single stem cell pellet in hematopoietic differentiation and realize vascularization and bone tissue regeneration of the stem cell pellet.
The preparation method of the stem cell pellet with osteogenesis and angiogenesis differentiation provided by the invention can promote the whole cell activity of the stem cell pellet, solve the defects of reduced cell activity and short maintenance time during in vitro induction of single stem cells, prolong the in vitro culture time of the stem cell pellet, effectively solve the key clinical problems of lack of vascular network, early necrosis after transplantation, low bone regeneration efficiency and the like of the stem cell pellet in the bone tissue engineering research application at present, and provide theoretical basis and experimental basis for the design of bone tissue engineering biological materials based on the stem cell pellet.
Drawings
FIG. 1 is a graph showing the characterization and multipotent differentiation capacity of sGMSCs; wherein a is GMSCs (pGMSCs) before MACS magnetic bead sorting and sGMSCs (Scale bar 50 μm) after STRO1 enrichment sorting; b is the expression condition of the sGMSCs surface marker under Isotype control; c is the osteogenic differentiation alizarin red S staining (Scale bar 500 μm) of pGMSCs and sGMSCs, and d is the adipogenic differentiation oil red O staining (Scale bar 100 μm) of pGMSCs and sGMSCs.
FIG. 2 shows the formation and morphology of sGMSC/HUVEC spheres (GHS); wherein a is the morphology of sGMSC sphere (GS), HUVEC Sphere (HS) and GHS (Scale bar 200 μm); b is that GHS has formed mature, regular spheres (Scale bar 400 μm) at 48h after co-cultivation; c is osteoinduction day 7 by immunofluorescence labeling of cell signature markers and labeling of nuclei with DAPI (cyan), sGMSCs in GHS (STRO 1 + ，CD31 - Yellow) and HUVECs (STRO 1 - ，CD31 + Magenta) fluorescent signal was evenly distributed, demonstrating good co-culture fusion of the two cells (scale bar 200 μm).
FIG. 3 is a graph showing HUVECs promoting stem cell activity and maintaining sphere stability in GHS system; wherein, a is GHS osteogenesis induced on day 7 of different cell proportion, compared with single sGMSC cell sphere, with the increase of HUVECs cell proportion, the cell activity in GHS system is improved, when GHR=1, the cell activity is highest, and there is statistical difference between groups. * Represents p < 0.05, p < 0.01, p < 0.001; b is a local enlarged graph of Live/Dead cell staining (green: living cells; red: dead cells) on day 7 of the osteogenesis of GS and GHS, punctiform Dead cells (yellow corner mark) appear at the core of GHS, the intercellular arrangement is compact, the edges of the cell sphere are clear, more Dead cells appear inside GS, the intercellular arrangement becomes loose, and the edges of the sphere are more blurred (Scale bar 200 μm).
FIG. 4 shows maintenance time of cellular activity in GHS and GS systems; wherein, a-b are respectively observed changes of cell activities of GS and GHS (scale bar 200 μm) in the period of 13 days of osteogenesis induction, and the proportion of two groups of dead cells is increased along with the extension of induction time, but compared with GS, the proportion of dead cells of GHS on the same time node is less, the morphology of cell spheres is well maintained, the edges are clearer, and the cells are more compact; c-d are Mean gray value visualizations of staining fluorescence signals of GS and GHS Live/read cells, respectively, with the proportion of GS viable cells at day 13 of the end of the observation being about 63.54 + -6.53% and GHS being about 79.05+ -1.97%; the proportion of dead cells of GS is about 35.87 ±3.93%, while GHS is about 21.28±2.27%; the proportion of dead cells of GHS was significantly lower than GS at this time (t= 5.593, representing p=0.005 < 0.05).
FIG. 5 is a graph showing the ability of HUVEC to promote osteogenic differentiation of the GHS system; wherein a-c are respectively HUVEC introduced co-culture to increase expression of GHS osteogenic differentiation genes ALP, RUNX2 and OCN on mRNA level; d-g are western blots respectively, and the increased expression of ALP, RUNX2 and OCN on protein level is also verified, wherein ns represents non-signaficant, p is less than 0.05, p is less than 0.01, and p is less than 0.001.
FIG. 6 shows that GHS constructed by HUVECs successfully compensates for the deficiency of GS in angiogenic differentiation; wherein a-b are the expression of the angiogenic differentiation genes CD31 and VEGF, respectively, of different GHS, and in GS, the angiogenic differentiation genes CD31 and VEGF are hardly expressed (the GHS group is deleted for comparing the relative expression amount between the groups); however, GHS introduced into the HUVECs construct starts to express angiogenic differentiation genes CD31 and VEGF, and as the proportion of HUVECs increases, the expression amount thereof at the mRNA level increases; c-e are Western blotting results of CD31 and VEGF respectively, and show that the expression trend of CD31 and VEGF on protein level is basically consistent with mRNA; in the figure, ns represents non-signalizing, p < 0.05, p < 0.01, and p < 0.001.
Detailed Description
The invention will now be described in detail with reference to specific examples, without limiting the scope of the invention.
The materials, reagents and the like used in this example are commercially available materials and reagents unless otherwise specified.
The cell culture technique in the following examples is a conventional technique, and the cell culture conditions are constant temperature of 37℃and carbon dioxide CO 2 The concentration was 5%.
English shorthand and Chinese meanings related in the following examples are as follows:
preparation of a maintenance medium: sGMSCs maintenance medium was α -MEM (Gibco Inc.) +1% P/S+10% FBS+1% Glutamax; HUVECs maintenance medium was EGM2 (Lonza Inc.) +1% P/S+10% FBS+1% Glutamax.
Preparation of differentiation induction culture medium for sGMSCs multidirectional differentiation experiment: the osteogenic induction differentiation culture medium is alpha-MEM+1% P/S+10% FBS+1% Glutamax+100 mu M L-ascorbic acid+10 nM dexamethasone+10 mM beta-sodium glycerophosphate; the adipogenic differentiation medium was alpha-MEM+1% P/S+1nM dexamethasone+10. Mu.g/mL insulin+100. Mu.M indomethacin+0.5 mM IBMX.
Stem cell pellet (GS) and vascularized stem cell pellet (GHS) osteogenic induction medium formulation: stem cell pellet osteogenesis induction medium is alpha-MEM+1% P/S+10% FBS+1% Glutamax+100 mu M L-ascorbic acid+10 nM dexamethasone+10 mM sodium beta-glycerophosphate. The vascularized stem cell pellet osteogenesis induction medium is EGM 2/alpha-MEM+1% P/S+10% FBS+1% GlutaMAX+100 mu M L-ascorbic acid+10 nM dexamethasone+10 mM sodium beta-glycerophosphate; culturing and amplifying the mixed cells by using corresponding maintenance culture mediums respectively, wherein the proportion (volume ratio) of a basic culture medium in the vascularized stem cell pellet osteogenesis induction culture medium is the same as the proportion of the number of the cells;
when sGMSCs: hucecs=5:1 (ghr=5), α -MEM: egm2=5:1 (i.e., α -MEM/egm2=5);
when sGMSCs: hucecs=4:1 (ghr=4), α -MEM: egm2=4:1 (i.e., α -MEM/egm2=4);
when sGMSCs: hucecs=3:1 (ghr=3), α -MEM: egm2=3:1 (i.e., α -MEM/egm2=3);
when sGMSCs: hucecs=2:1 (ghr=2), α -MEM: egm2=2: 1 (i.e. α -MEM/egm2=2);
when sGMSCs: hucecs=1:1 (ghr=1), α -MEM: egm2=1:1 (i.e., α -MEM/egm2=1).
Preparation of MACS magnetic bead sorting buffer: 0.5% BSA+2mM EDTA, 250mg BSA powder, 200. Mu.L of 0.5M EDTA solution were weighed and mixed into 1 XDBS solution in a final volume of 50mL.
Reagents and consumables used for sGMSCs sorting and enrichment include: MACS magnetic bead sorting buffer, PE-coupled rabbit anti-human STRO1 antibody (Abcam), anti-PE immune sorting magnetic beads (Miltenyi), magnetic bead sorting magnetic frame (Miltenyi), MS magnetic bead sorting column (Miltenyi), 6-well plate, 0.25% pancreatin, DPBS solution, sGMSCs maintenance medium
Example 1
In this example, sGMSCs with higher differentiation potential are obtained through sorting and enrichment, and the specific process is as follows:
(1) The P1-2 GMSCs (gingival mesenchymal stem cells, purchased from Cyagen Biosciences Inc) were subjected to DPBS washing and 0.25% pancreatin digestion, resuspended in 1mL of MACS magnetic bead sorting buffer pre-cooled at 4deg.C and counted, and the total cell number was not less than 1×10 7 And each.
(2) After counting, the supernatant was centrifuged off, 50. Mu.L of MACS magnetic bead sorting buffer containing PE-conjugated rabbit anti-human STRO1 antibody (final concentration 5. Mu.g/mL) was added for resuspension, incubation was carried out at 4℃for 10min in the dark, after antibody incubation was completed, the supernatant was centrifuged off, and unbound antibody was washed off by adding MACS magnetic bead sorting buffer.
(3) After washing the residual antibody, anti-PE immune sorting magnetic beads (20 mu L/10) are added 7 Individual cells), incubation at 4℃for 30min in the absence of light.
(4) And assembling a magnetic bead separation magnetic frame and an MS magnetic bead separation column, and adding 100 mu L MACS magnetic bead separation buffer solution wetting column after the temperature of the MS magnetic bead separation column is restored.
(5) And (3) adding MACS magnetic bead sorting buffer solution into the cell-magnetic bead mixed solution for further resuspension, then slowly injecting into an MS magnetic bead sorting column for magnetic separation, and collecting separated positive cells, namely sGMSCs (STRO 1 positive human gingival mesenchymal stem cells).
(6) The positive cell suspension was centrifuged and the supernatant was discarded, resuspended in sGMSCs maintenance medium, and plated on 6-well plates or used directly in subsequent experiments.
Characterizing the sGMSCs obtained by screening, which concretely comprises the following steps:
1) Morphological characteristics of sGMSCs
Inoculating sGMSCs after separation and enrichment and GMSCs before separation into 6-hole plates respectively, wherein each hole is inoculated with 2×10 cells 5 Next day, adherent cell morphology was observed under an inverted microscope.
2) sGMSCs surface marker expression
Cell count was performed after magnetic bead sorting, and 2X 10 5 The single cell suspension of STRO1+GMSC after individual bead sorting (i.e. sGMSCs after enrichment) is added into a flow tube. Fluorescence labelled CD14, CD31, CD34, CD45, CD73, CD90, CD105, CD146 antibodies (all purchased from abcam) were added according to the manufacturer's instructions, while rabbit anti-human IgG antibodies (abcam) were selected as negative controls. After incubation at room temperature for 2h in the absence of light, cells were washed 3 times with PBS to remove unbound antibody. Cells were resuspended at room temperature and loaded for detection. After the flow cytometer (Beckman) has set the electronic parameters of the instrument and adjusted for compensation, negative controls are used to define negative boundaries and to detect the fluorescence intensity of the cells and the number of positive cells.
3) Multi-directional differentiation potential detection of sGMSCs, including osteogenic and adipogenic differentiation performance detection
And (3) osteogenic differentiation detection: GMSC not subjected to magnetic bead sorting enrichment was used as a control, and the density was 1X 10 5 The sGMSC and GMSC of each well were inoculated into a 12-well plate containing a GMSCS maintenance medium. After 72h the osteogenic induction medium (α -MEM+1% P/S+10% FBS+1% Glutamax+100 μ M L-ascorbic acid+10 nM dexamethasone+10 mM sodium β -glycerophosphate) was changed, once every 48 h. After 21 days of continuous induction, the cells were washed 3 times with PBS and then with alizarin at room temperaturePlain red S dye liquor (Solarbio) for 1h. After rinsing, the osteogenesis was observed using an inverted microscope.
Adipogenic differentiation assay: after 3 days of inoculation and maintenance, the cells were washed 3 times with PBS after continuous induction for 14 days and then fixed with 4% PFA (Biosharp) for 15min after 3 days of replacement with lipid induction medium (. Alpha. -MEM+1% P/S+1nM dexamethasone+10. Mu.g/mL insulin+100. Mu.M indomethacin+0.5 mM IBMX). Cells were stained with oil red O (Solarbio) stain at room temperature according to the manufacturer's instructions. After rinsing, the lipid droplets were observed under an inverted microscope.
Characterization and multidirectional differentiation capacity of sGMSCs are shown in figure 1, a is that GMSCs before MACS magnetic bead sorting and STRO1 are enriched in sGMSCs after sorting, and in terms of cell morphology, the single-layer growth of the GMSCs before sorting takes on a long fusiform shape. After immunomagnetic bead sorting, sGMSCs show a polygonal shape or a long fusiform shape, and more pseudo feet (scale bar 50 μm) are protruded to the periphery, so that the sGMSCs can be amplified to reach the pooling rate of 80-90% within 3 days. In fig. 1 b is the expression of the surface markers of the gmscs in the Isotype control, the sorted cell subsets almost all expressed CD73 (98.88%), CD90 (99.86%), CD105 (99.76%), and highly expressed progenitor cell markers STRO1 (87.91%), almost not CD14 (0.05%), CD31 (0.07%), CD34 (0.06%) and CD45 (0.64%), confirming that the gmscs are a population of mesenchymal stem cells. In FIG. 1 c is osteogenic differentiated alizarin red S staining of GMSCs and sGMSCs. In FIG. 1, d is the lipid-forming differentiation oil red O staining of GMSCs and sGMSCs, it can be seen that sGMSCs show better potency than GMSCs in terms of osteogenesis and lipid-forming differentiation.
Example 2
A method of constructing vascularized stem cell spheres for bone regeneration, comprising the steps of:
(1) After sGMSCs (obtained by screening and enriching in example 1) and HUVECs (umbilical vein endothelial cells, purchased from Cyagen Biosciences Inc) were digested with 0.25% pancreatin into single cell suspensions, the cell numbers per unit volume were calculated;
(2) Calculating corresponding cell suspension volume and corresponding culture medium ratio according to the cell composition ratio:
sGMSCs/HUVECs=5 (i.esGMSCs cell number of 8.33X10 5 HUVECs of 1.67×10 5 Individual). alpha-MEM/egm2=5 in osteoinductive medium;
(3) According to the experimental grouping requirement, the ratio calculated in the step (2) is used for obtaining a mixed culture medium (alpha-MEM/EGM2+%P/S+10%FBS+1% Glutamax+100 mu M L-ascorbic acid+10 nM dexamethasone+10 mM sodium beta-glycerophosphate), and part of the mixed culture medium is added into a 6-hole ultra-low adhesion cell culture plate to infiltrate the bottom of the plate in advance, wherein the volume of the mixed culture medium added into each hole is 1.5mL; meanwhile, calculating the volume of the required cell suspension according to the step (2), uniformly mixing the two cells according to the inoculation proportion, and removing the supernatant after centrifugation;
(4) Adding 1mL of mixed culture medium which is proportioned in advance into the mixed cell sediment obtained in the step (3), re-suspending, inoculating into a 6-hole ultralow-adhesion cell culture plate, ensuring the final volume of each hole of culture medium to be 2.5mL, and culturing to obtain vascularized stem cell spheres or stem cell spheres (the vascularized stem cell spheres can be formed into more regular cell spheres after 48 hours);
(5) According to experimental requirements, the vascularized stem cell pellet or stem cell pellet obtained in the step (4) can be continuously cultured, and a half-amount liquid exchange and fresh culture medium exchange are carried out every 48 hours.
Example 3
A method of constructing vascularized stem cell spheres for bone regeneration, differing from example 2 only in that: sGMSCs/HUVECs=4 (i.e. sGMSCs cell number is 8.00×10) 5 HUVECs are 2.00×10 5 And a) in the osteogenic induction medium α -MEM/egm2=4.
Example 4
A method of constructing vascularized stem cell spheres for bone regeneration, differing from example 2 only in that: sGMSCs/HUVECs=3 (i.e. sGMSCs cell number is 7.50X10) 5 HUVECs were 2.50X10 5 And a) in the osteogenic induction medium α -MEM/egm2=3.
Example 5
A method of constructing vascularized stem cell spheres for bone regeneration, differing from example 2 only in that: sGMSCs/HUVECs=2 (i.e. sGMSCs cell number is 6.67×10) 5 HUVECs 3.33X10 5 And) osteogenesis inducing culturealpha-MEM/egm2=2 in the medium.
Example 6
A method of constructing vascularized stem cell spheres for bone regeneration, differing from example 2 only in that: sGMSCs/HUVECs=1 (i.e. sGMSCs cell number 5.00×10) 5 HUVECs are 5.00×10 5 And a) in the osteogenic induction medium α -MEM/egm2=1.
Comparative example 1
The construction method of stem cell pellet is different from example 2 only in that only sGMSCs are inoculated, i.e., the number of sGMSCs cells is 1.00×10 6 In addition, alpha-MEM based osteoinductive media was not added with EGM2.
Comparative example 2
The construction of endothelial cell pellets differs from example 2 only in that HUVECs are only seeded, i.e.the number of HUVECs cells is 1.00×10 6 In addition, no alpha-MEM was added to EGM 2-based osteoinductive medium.
Effect examples
1. Morphology and internal cell fusion of the vascularized Stem cell pellet obtained in example 6
The method comprises the following steps:
step 1: sphere formation was observed using an inverted microscope at four time nodes 0, 12, 24, 48h of vascularized stem cell sphere culture. Selecting a sample on day 7 to be subjected to immunofluorescence staining, and separating liquid under the condition of low-rotation-speed short-time centrifugation (300 rpm,1 min) to avoid dissociation of spheres;
step 2: harvesting the suspended stem cell balls into a centrifuge tube, adding DPBS, and rinsing on a shaking table for 3 times;
step 3: fixation with 4% PFA was performed for 2h. After fixation PFA was blotted off and rinsed 3 times with DPBS;
step 4: degreasing and decoloring the cell balls for 12h by using a tissue transparent agent CUBIC-L after fixation;
step 5: immediately after rinsing, blocking with 3% BSA for 2h at room temperature;
step 6: after blocking, a universal antibody dilution (Sigma Aldrich) containing 1:100 monoclonal rabbit anti-human CD31 primary antibody (abcam) and 1:200 monoclonal mouse anti-human STRO1 primary antibody (Santa cruz) was added and incubated overnight at 4 ℃;
step 7: the following day, alexa Fluor 594-labeled goat anti-rabbit secondary antibody (abcam) and Alexa Fluor 488-labeled goat anti-mouse secondary antibody (abcam) were added and incubated separately for 1h at room temperature in the dark. PBST was rinsed 3 times away from light, and the nuclei were counterstained with 1:1000DAPI (Solarbio) and observed for STRO1 and CD31 distribution using a laser confocal microscope (Leica).
The results are shown in FIG. 2, in which FIG. 2 a shows that under ultra-low adhesion plate conditions, GHS, GS and HS all initially formed three-dimensional spheres within 24 hours, with no difference in morphology formation time (scale bar 200 μm). Three groups of spheres differ in morphology and diameter: the GS edge is smooth, and the diameter is smaller, which is about 374.32 +/-68.91 mu m; HS is spindle-shaped, has more cell protrusions at the edge and larger diameter, and is about 637.39 +/-130.22 mu m; the sphere formed by GHS is more regular, with a small number of cell protrusions still visible at the edges, probably HUVECs that have not yet completed contact, with a diameter of about 563.63 ±61.09 μm. FIG. 2 b shows that at 48h of co-cultivation most of the GHS, GS and HS morphologies were mature, forming a number of edge-smoothed spheres (scale bar 400 μm).
To determine the cellular distribution of sGMSC and HUVEC within GHS, the inventors performed a double-labeled immunofluorescent staining assay of STRO1 and CD31 on day 7 of osteogenic induction of GS and GHS. The results show that: in GS, STRO1 positive sgscs are evenly distributed, whereas CD31 fluorescent signal is negative since the sgscs do not express CD 31; in HS, CD31 positive HUVECs are evenly distributed, whereas STRO1 fluorescent signal is negative since HUVECs do not express STRO1. However, in GHS, the fluorescence signals of the gmsc highly expressing STRO1 and the HUVEC highly expressing CD31 were evenly distributed and exhibited an approximately spherical morphology (c in fig. 2, scale bar 200 μm), which further confirmed the effectiveness of fusion of the gmsc and HUVEC to spheres in three-dimensional co-culture.
2. Detecting the cell activity in vascularized stem cell ball system comprising different cell ratios
The method comprises the following steps:
(1) Monitoring of cell Activity in vascularized Stem cell pellet System
Step 1: digestion, counting and mixing of the sGMSC and HUVEC (set GS and GHS groups, where GHR=5, 4, 3, 2 or 1) resulted in a total volume of 100. Mu.L of single cell suspension;
step 2: at 10 4 Total cell number density of wells they were seeded in ultra low adhesion 96 well plates (Corning Elplasia), 3 multiplex wells were set per group;
step 3: after induction in osteogenic induction medium for 7 days, the plates were removed and allowed to return to room temperature;
step 4: using CellTiter-Lumi TM Steady Plus Luminescent Cell Viability Assay Kit (Beyotime) assay for ATP content of cell pellets per well: 100. Mu.L CellTiter-Lumi was added TM Working solution is vibrated for 2-5 min at room temperature to fully lyse cell balls, and incubation is continued for 10min at room temperature to ensure stable luminous signals;
step 5: after incubation, the chemiluminescent intensities of each group were measured using a fluorescent microplate reader (Tecan).
The results show that changing the ratio of HUVEC in GHS (ghr=5, 4, 3, 2, 1) was osteoinductive for 7 days, and that the introduction of HUVEC was shown to enhance cellular activity in GHS system by detection of ATP by chemiluminescence, with statistical differences between the different groups, and with increasing ratio of HUVEC, the higher the activity of GHS system. When ghr=1, the cell viability in the GHS system is significantly higher than GS (a, P < 0.05, P < 0.01, P < 0.001 in fig. 3). 3. Detection of maintenance of cell activity in vascularized stem cell pellet system composed of different cell ratios
Step 1: at 10 4 Total cell number density per well they were seeded in ultra low adhesion 96 well plates (Corning Elplasia) to construct GS and GHS (ghr=1) respectively, with 3 multiplex wells per group;
step 2: on days 1, 3, 5, 7, 9, 11, 13 of its osteogenesis induction, staining was performed using the Calcein/PI Assay Kit (Beyotime);
step 3: imaging under a laser confocal microscope and recording GS and GHS in the time node in the step 2;
step 4: live-dead cell fluorescence intensity of step 3 images was quantified with Image J and plotted using software GraphPad prism 9.0.
The results showed that the GS and GHSI viable and dead cell staining results on day 7 of culture showed: although there was a small number of cell deaths in both GS and GHS systems, dead cells (red) in GS were significantly more than in the contemporaneous GHS. GHS only presents a small number of dead cells (yellow triangular marks) in the inner core area of the sphere, the cells are closely arranged, the edges of the cell sphere are clear, and the cell death starts from the sphere core and gradually expands to the periphery of the sphere; more dead cells appeared inside the GS, the intercellular arrangement became loose and the edges of the spheres were more blurred (b, scale bar 200 μm in fig. 3).
GS on day 3 of culture, the proportion of viable cells was already below 90%, and punctiform dead cells began to appear in the sphere core region; over the induction time, the number of dead cells gradually increased and the morphology of the spheres changed, and the edges and intercellular became blurred (fig. 4 a, scale bar 200 μm). At day 13, about 35.87.+ -. 3.93% of the cells within the GS system had died, while the living cell fraction was about 63.54.+ -. 6.53% (c in FIG. 4).
In contrast, GHS began to appear punctate dead cells in the sphere core region on days 5-7 of culture (b in fig. 4, scale bar 200 μm). Although the number of dead cells also gradually increased over time, the proportion of viable cells began to be less than 90% on days 9-11 of osteoinduction (d, scale bar 200 μm in FIG. 4). GHS is still able to maintain good spheroid morphology and intercellular compact and clear characteristics by day 13, with viable cell ratio of about 79.05+ -1.97%; and the number of dead cells at this time was about 21.28±2.27%, significantly less than GS at the same time point (t= 5.593, =p=0.005 < 0.05).
The results of fig. 4 further demonstrate the promoting effect of HUVEC on cellular activity in GHS systems, and also suggest that intercellular interactions of the gmsc with HUVEC in GHS systems may contribute to long term stabilization of the spheroid system.
4. Detection of angiogenic differentiation markers of stem cell spheroids
The method comprises the following steps:
step 1: vascularized stem cell pellets (ghr=5, 4, 3, 2, 1) were selected for each cell composition ratio after 7 days osteoinduction.
Step 2: and (3) extracting RNA and total protein of each group of samples in the step (1) to finish the preparation of the early-stage samples of qRT-PCR and Western Blot.
Step 3: using the samples of step 2, the gene and protein expression amounts of the osteogenic differentiation markers OCN, RUNX2, ALP, and the angiogenic differentiation markers CD31, VEGF were examined for each group of samples by qRT-PCR and Western Blot experiment.
The results showed that mRNA expression levels of osteogenic genes including OCN, RUNX2, ALP were significantly increased in each GHS group co-cultured with HUVEC after 7 days of osteogenic induction, and there was a statistical difference from the GS group. Furthermore, with increasing HUVEC cell proportion in GHS system (i.e. increasing GHR), the expression levels of OCN, RUNX2, ALP were significantly increased. mRNA expression levels of osteogenic related genes OCN, RUNX2, ALP were highest when the ratio of gmsc to HUVEC cells within the GHS system was 1:1 (i.e. ghr=1) (fig. 5 a-c). In FIG. 5, d is a western blot of GS and GHS of each group, and e-g in FIG. 5 were normalized and statistically analyzed, showing that as GHR increases, the relative protein expression levels of OCN, RUNX2 and ALP also increase, which showed a trend consistent with the mRNA expression levels.
To determine whether different proportions of HUVECs could promote angiogenic differentiation in the GHS system, mRNA expression levels of the angiogenic differentiation genes of each group of GHS, including CD31 and VEGF, were further examined after 7 days of osteoinduction. The results show that the mRNA expression levels of CD31 and VEGF for GHS were significantly higher than other ratios of GHS (i.e. ghr=5, 4, 3, 2) when ghr=1 (fig. 6 a-b). The normalized quantitative statistics of d-e in FIG. 6 also confirm that the overall trend of CD31 and VEGD relative protein expression is consistent with mRNA expression levels in the Western blot of FIG. 6.
Taken together, the results in FIG. 5 demonstrate that co-culture of HUVECs with sGMSCs may promote an increase in the osteogenic differentiation capacity of the sGMSCs. The results in fig. 6 suggest that with increasing proportion of co-cultured HUVECs, the angiogenic differentiation capacity of GHS increases, contributing to early vascularization of GHS and maintaining long-term stability.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The stem cell pellet is obtained by three-dimensional co-culture of STRO1 positive gingival mesenchymal stem cells and HUVECs.
2. The stem cell pellet of claim 1 wherein the STRO1 positive gingival mesenchymal stem cells highly express mesenchymal stem cell markers CD73, CD90 and CD105 and highly express progenitor cell marker STRO1; and/or the ratio of the STRO1 positive gingival mesenchymal stem cells to the cells of the HUVEC is (1-6): 1.
3. The stem cell pellet of claim 2, wherein the stem cell pellet highly expresses vascularization markers CD31 and VEGF, and highly expresses osteogenic differentiation markers RUNX2 and OCN; and/or the diameter of the stem cell sphere is 500-700 μm.
4. A method for preparing the stem cell pellet as claimed in any one of claims 1 to 3, comprising the steps of:
mixing the gingival mesenchymal stem cells positive to STRO1 and HUVEC, and culturing in an induction culture medium to obtain the stem cell pellet.
5. The preparation method according to claim 4, wherein the STRO1 positive gingival mesenchymal stem cells and HUVECs are mixed in a cell ratio of (1-6): 1.
6. The method of claim 4, wherein the induction medium comprises EGM2, α -MEM, FBS, glutaMAX, L-ascorbic acid, dexamethasone, and sodium β -glycerophosphate.
7. The method according to claim 6, wherein the volume ratio of the alpha-MEM to the EGM2 in the induction medium is (1-6): 1.
8. The method according to any one of claims 4 to 7, wherein the time of the cultivation is 1 to 14 days.
9. Use of the stem cell pellet of any one of claims 1 to 3 or the preparation method of any one of claims 4 to 8 in any one of (a 1) to (a 6):
(a1) Preparing vascularized tissue;
(a2) Preparing a product for promoting bone tissue repair/regeneration;
(a3) Preparing an angiogenesis promoting product;
(a4) Screening for anti-angiogenic/pro-angiogenic drugs;
(a5) The cell activity of stem cells in vitro induction is improved;
(a6) Prolonging the cell activity time of stem cells in vitro.
10. A vascularized tissue comprising stem cell pellets according to any of claims 1-3 after induction culture.
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